High fructose corn syrup (HFCS) is a processed form of corn syrup having a high fructose content and a sweetness comparable to sugar, making HFCS useful as a sugar substitute in soft drinks and other processed foods. HFCS currently represents a billion dollar industry. The process of producing HFCS has progressed over the years from acid hydrolysis to a sequence of enzyme-catalyzed reactions:
(1) Liquefaction: α-Amylases (EC 3.2.1.1) are first used to degrade a starch suspension containing 30-40% w/w dry solids (ds) to maltodextrans. α-Amylases are endohydrolases that catalyze the random cleavage of internal α-1,4-D-glucosidic bonds. Because liquefaction typically is conducted at high temperatures, e.g., 90-100° C., thermostable α-amylases, such as an α-amylase from Bacillus sp., are preferred for this step.
(2) Saccharification: Glucoamylases and/or maltogenic α-amylases commonly are used to catalyze the hydrolysis of non-reducing ends of the maltodextrans formed after liquefaction, releasing D-glucose, maltose and isomaltose. De-branching enzymes, such as pullulanase, can be used to aid saccharification. Saccharification typically takes place under acidic conditions at elevated temperatures, e.g., 60° C., pH 4.3. Glucoamylases used in this process typically are obtained from fungi, e.g., Aspergillus niger glucoamylase (AnGA) used in Optidex® L400 or Humicola grisea glucoamylase (HgGA). Maltogenic α-amylases currently used for this application include plant amylases and the α-amylase from Aspergillus oryzae, the active ingredient of Clarase® L. Saccharification can be used to produce either high-maltose or glucose-rich syrups.
(3) Isomerization: A glucose-rich syrup can be processed further to produce fructose, when sweeter products are desired. Isomerization of glucose to fructose is catalyzed by glucose isomerase and yields about 42% (w/v) fructose, 50-52% glucose, and a mixture of other sugars. Additional manipulations ultimately can yield commercial grade HFCS having a fructose content of 42%, 55%, or 90%, for example.
The α-amylases and glucoamylases are added directly to a process batch of corn syrup and are not reused. Glucose isomerases, on the other hand, are immobilized on columns over which the sugar mixture is passed. The glucose isomerase columns are reused until the enzymes lose most of their activity.
The saccharification step is the rate-limiting step of HFCS production. Saccharification typically occurs over 48-72 hours, by which time many fungal glucoamylases have lost significant activity. Further, although maltogenic α-amylases and glucoamylases both can be used to catalyze saccharification, the enzymes typically operate at different optimal pH and temperatures. For example, maltogenic α-amylases typically have a pH optimum of at least pH 5.0 and a temperature optimum of less than 55° C., while AnGA typically has a pH optimum of pH 4.0-4.5 and a temperature optimum of about 60° C. The difference in reaction conditions between the two enzymes necessitates adjusting the pH and temperature, which slows down the overall the process and may give rise to the formation of insoluble amylose aggregates. Any remaining bacterial α-amylase will be inactivated when the pH is lowered; however, the bacterial α-amylase may be replaced later by an acid-stable α-amylase.
Ideally, the saccharification step yields a syrup with a composition of about 95-97% w/w glucose, 1-2% w/w maltose, and 0.5-2% w/w isomaltose. This glucose-rich syrup either can be used in the isomerization reaction, step (3) above, or used for the production of crystalline glucose. These high glucose concentrations are not easily achieved. For example, Trichoderma reesei glucoamylase (TrGA) offers improved specific activity relative to AnGA or HgGA; however, TrGA yields a product having a final glucose concentration typically about 88% w/v. Further, high glucose concentrations in the syrup promote the conversion of glucose to maltose and maltotriose.
Accordingly, there is a need in the art for an improved process of making HFCS, which includes a saccharification step that uses an α-amylase with a pH optimum and temperature optimum compatible with the use of fungal glucoamylases. There is also a need for an α-amylase that can catalyze saccharification in less time. Further, there is a need for an α-amylase that can accomplish these objectives, while producing a syrup after saccharification that has a glucose concentration of about 96% w/w.